This proposal addresses broad Challenge Area (04) Clinical Research and specific Challenge topic, 04- HL-103: Assess the role of leukocyte interaction with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases. The cadherin Neural Cell Adhesion Molecule (NCAM) is key in leukocyte adhesion to endothelial cells. Upon signaling by chemokines such as MCP1 leukocytes are drawn toward endothelial cells. Appositional interactions ("zippering") between NCAM molecules from the leukocyte and endothelial cell forms a tether between them and allows the leukocyte's cell surface antigens to interact with those of the endothelial cell. This is followed by cell:cell interactions such as diapedesis. The resulting sub-endothelial inflammatory processes can result in atheroma formation and atherosclerosis. However, this sequence may be blocked by the insertion of the NCAM by sialic acid decorated isoforms termed polysialated NCAM (PSA-NCAM). These have poly hydrated [unreadable]2,8 sialic acid chains in the extracellular domain that interfere with the zipper adhesion to NCAM. We have documented the presence of PSA-NCAM in the glycocalyx of human vascular endothelial cells in arteries, veins and lymphatics of all organs (10 organ systems) studied. We also have shown that expression of the two required polysialyltransferases (ST8Sia II/STX and ST8Sia IV/PST) is estrogen receptor-mediated. Thus, the enzymatic replacement of NCAM with PSA-NCAM depletes the parent NCAM molecules, so that the adhesion (NCAM):non- adhesion (PSA-NCAM) balance is highly leveraged. In the case of the vascular endothelium, the result may be minimization of leukocyte adhesion and sub endothelial inflammation, which would diminish the formation of atheromas and atherosclerosis. In previous studies we have shown that estradiol induces PST and STX in rat brain tissue and increases PSA- NCAM at the expense of NCAM in human umbilical vein endothelial cells (HUVEC). The estrogen receptor blocker fulvestrant had the opposite effect. In this proposal, using cardiovascular-relevant cells [human leukocytes (THP1) and human arterial endothelial cells (HCAEC)] we will examine estrogen-regulated leukocyte adhesion: We will determine the NCAM:PSA-NCAM ratio induced by estradiol and fulvestrant, their effects on sialylation enzymes and their effect on leukocyte-endothelial adhesion. We will compare the results with effects of the Ras pathway inducer valproic acid which is known to induce PSA-NCAM. Further, the effects of siRNA inhibition of STX and PST on the NCAM:PSA-NCAM balance and on leukocyte adhesion to endothelial cells will also be investigated. In this manner, we will test whether estrogen's cardioprotective effects could be due to increased NCAM sialylation, leading to decreased leukocyte adhesion and sub-endothelial inflammation. Impact: Heart disease is the single greatest cause of death and results in staggering healthcare expenditures and personal suffering. Finding new ways to protect against atherosclerosis is imperative for the health of the nation. Because of its importance to atherogenesis, understanding of the regulation of leukocyte-endothelial cell interactions is of the greatest clinical importance. This proposal will test the role of estrogen in preventing leukocyte adhesion that is a premonitory step toward atheroma formation. Such action would be consistent with the well- documented cardio-protective effects of estrogen in experimental and clinical studies. If this proposal is successful in exposing the mechanism by which estrogen affects leukocyte adhesion to endothelial cells it may be possible to alter already available treatments to avoid atherosclerosis and develop novel preventative treatments specifically aimed at regulating NCAM sialylation in clinical cardioprotection. Arteriosclerotic heart disease, the number one killer in the USA, starts from blood vessel inflammation that ultimately puts the individual at risk of a heart attack. This proposal will explore whether estrogen protects against atherosclerosis through production of specific, estrogen-regulated enzymes that inhibit inflammatory cells from entering into blood vessels. This proposal may improve the application of anti-atherosclerosis agents and lead to development of new agents to protect blood vessels from atherosclerosis and its complications. PUBLIC HEALTH RELEVANCE: Arteriosclerotic heart disease, the number one killer in the USA, starts from blood vessel inflammation that ultimately puts the individual at risk of a heart attack. This proposal will explore whether estrogen protects against atherosclerosis through production of specific, estrogen-regulated enzymes that inhibit inflammatory cells from entering into blood vessels. This proposal may improve the application of anti-atherosclerosis agents and lead to development of new agents to protect blood vessels from atherosclerosis and its complications.